The long range goal of this project is to determine the molecular details of DNA chain initiation in the yeast S. cerevisiae by identifying the proteins that catalyze and control the process, isolating them, and determining how they work. Two approaches are being pursued: the direct isolation of such proteins from extracts of the yeast cell using biochemical assays specific for DNA chain initiation and elongation, and an indirect approach of isolating temperature-sensitive mutants of yeast defective in DNA synthesis to identify essential gene products. A yeast DNA primase capable of DNA chain initiation has been purified in a complex with the major yeast DNA polymerase. Free DNA primase has also been purified. Both synthesize short discrete length RNA primers that can be extended by DNA polymerase. The subunit composition and subunit functions of both proteins will be determined. The proteins will be subjected to additional fractionation, neutralizing and binding monoclonal antibodies will be isolated, and the subunit structural genes will be isolated. The specific antibody probes and isolated genes will be used to determine the relationship between the free DNA primase and the primase in the polymerase-primase complex. The mechanism of concerted DNA priming and DNA chain elongation will be examined in detail. These studies will provide a detailed model for DNA chain initiation and elongation events at chromosomal replication forks in the eukaryotic cell.